Significant structural differences exist between the O-glycosylation pathways and patterns of lower eukaryotes (i.e., yeast and filamentous fungi) and mammals (particularly humans). Because the human immune system may recognize the alternative glycosylation of lower eukaryotes as foreign, any protein-based therapeutic products produced in fungal systems have the potential to provoke an immunogenic response when injected into humans. This response may limit the effectiveness of a therapeutic over multiple administrations and, in the most serious cases, may cause adverse effects in the patient.
In fact, the presence of fungal glycosylation is a common signal for clearance by the innate human immune system; see, e.g., Ballou, C. E., 1990 Methods Enzymol. 185:440. This raises concern about the potential for rapid clearance or immunogenicity of therapeutic proteins produced in yeast, such as Pichia pastoris and injected into humans, including but not limited to proteins having N and O glycans typical of yeast.
Previous attempts at reducing the immunogenicity of therapeutic proteins produced in yeast have largely focused on reducing the immunogenicity of N glycans (see, e.g., Gerngross U.S. Pat. No. 7,029,872). More recent attempts have focused on reducing or eliminating altogether fungal O-glycosylation in order to reduce or eliminate this response; see, e.g., Tanner, U.S. Pat. No. 5,714,377; and Bobrowicz et al., WO2007/061631. By contrast, the present inventors have surprisingly found unexpected advantages in producing a protein with certain O-glycosylation patterns similar to the O-glycosylation observed on native human glycoproteins, or closer to the O-glycosylation observed on recombinant glycoproteins produced in mammalian cells.